The esterification and modification of n-3 and n-6 polyunsaturated fatty acids by hepatocytes and liver microsomes of turbot (Scophthalmus maximus)

The present study was undertaken to establish whether the formation of 22:6n-3 from 18:3n-3 and/or 20:5n-3 can occur in turbot liver and if this conversion is consistent with the operation of a Delta4 desaturase-independent pathway. At the same, time the effects of feeding a diet devoid of long chain polyunsaturated fatty acids (PUFA) on the patterns of esterification and modification of 18:3n-3, 20:5n-3 and 18:2n-6 by turbot hepatocytes and liver microsomes were examined. For this purpose, two groups of fish (25-30 g) were employed: one was fed a commercial diet containing fish oil (FO) and thus rich in long chain n-3 PUFA and the other was fed an experimental diet based on olive oil (OO). After 5 months of feeding, hepatocytes and liver microsomes isolated from individuals in the two groups of fish were incubated with [1-(14)C]-PUFA [either 18:3n-3, 20:5n-3 or 18:2n-6]. After 3 h of incubation, most radioactivity from all three radiolabelled substrates incorporated into lipids by hepatocytes and microsomes was recovered in the original substrate. The formation of desaturation products of n-3 radiolabelled substrates was higher in hepatocytes isolated from OO-fed than FO-fed fish. Small amounts of radiolabelled 22:6n-3 were formed from [1-(14)C]18:3n-3 and [1-(14)C]20:5n-3, but only by hepatocytes from fish fed OO, which also synthesised a small amount of radiolabelled 24:6n-3 from 14C-20:5n-3. Elongation products predominated over desaturation products in hepatic microsomes from both groups of fish studied, particularly in microsomes from fish fed FO. The results confirm that regardless of the long chain PUFA content of the diet, the production of 22:6n-3 in turbot liver from 18:3n-3 and/or 20:5n-3, and of 20:4n-6 from 18:2n-6, is very limited. The presence of radiolabelled 24:6n-3 in microsomes coupled with the absence of radiolabelled 22:6n-3 suggests that the formation of 22:6n-3 that does occur in turbot liver cells, may involve C24 intermediates and peroxisomal beta-oxidation.     

The influence of dietary essential fatty acids on uterine C20 and C22 fatty acid composition.

The effect of dietary fatty acids on uterine fatty acid composition was studied in rats fed control diet or semi-synthetic diet supplemented with 1.5 microliter/g/day evening primrose oil (EPO) or fish oil (FO). Diet-related changes in uterine lipid were detected within 21 days. Changes of 2- to 20-fold were detected in the uterine n-6 and n-3 essential fatty acids (EFA) and in certain saturated and monounsaturated fatty acids. The FO diet was associated with higher uterine C20 and C22 n-3, and the EPO diet, with higher uterine n-6 fatty acid. High uterine C18:2 n-6 was detected in neutral lipid (NL) of rats fed high concentrations of this fatty acid, but there was little evidence of selective incorporation or retention of C18:2 n-6 by uterine NL. The incorporation of EFA into uterine phospholipids (PL) was greater than NL EFA incorporation, and uterine PL n-3/n-6 ratios showed greater diet dependence. Tissue/diet fatty acid ratios in NL and PL also indicated preferential incorporation/synthesis of C16:1 n-9, and C16:0, and there was greater incorporation of C12:0 and C14:0 into uteri of rats fed EPO and FO. Replacement of 50-60% of arachidonate with n-3 EFA in uterine PL may inhibit n-6 EFA metabolism necessary for uterine function at parturition.     

Fatty acid metabolism in Atlantic salmon (Salmo salar L.) hepatocytes and influence of dietary vegetable oil

Isolated hepatocytes from Atlantic salmon (Salmo salar), fed diets containing either 100% fish oil or a vegetable oil blend replacing 75% of the fish oil, were incubated with a range of seven (14)C-labelled fatty acids. The fatty acids were [1-(14)C]16:0, [1-(14)C]18:1n-9, 91-(14)C]18:2n-6, [1-(14)C]18:3n-3, [1-(14)C]20:4n-6, [1-(14)C]20:5n-3, and [1-(14)C]22:6n-3. After 2 h of incubation, the hepatocytes and medium were analysed for acid soluble products, incorporation into lipid classes, and hepatocytes for desaturation and elongation. Uptake into hepatocytes was highest with [1-(14)C]18:2n-6 and [1-(14)C]20:5n-3 and lowest with [1-(14)C]16:0. The highest recovery of radioactivity in the cells was found in triacylglycerols. Of the phospholipids, the highest recovery was found in phosphatidylcholine, with [1-(14)C]16:0 and [1-(14)C]22:6n-3 being the most prominent fatty acids. The rates of beta-oxidation were as follows: 20:4n-6>18:2n-6=16:0>18:1n-9>22:6n-3=18:3n-3=20:5n-3. Of the fatty acids taken up by the hepatocytes, [1-(14)C]16:0 and [1-(14)C]18:1n-9 were subsequently exported the most, with the majority of radioactivity recovered in phospholipids and triacylglycerols, respectively. The major products from desaturation and elongation were generally one cycle of elongation of the fatty acids. Diet had a clear effect on the overall lipid metabolism, with replacing 75% of the fish oil with vegetable oil resulting in decreased uptake of all fatty acids and reduced incorporation of fatty acids into cellular lipids, but increased beta-oxidation activity and higher recovery in products of desaturation and elongation of [1-(14)C]18:2n-6 and [1-(14)C]18:3n-3.     

Beta-oxidation of medium chain (C8-C14) fatty acids studied in isolated liver cells

The beta-oxidation and esterification of medium-chain fatty acids were studied in hepatocytes from fasted, fed and fructose-refed rats. The beta-oxidation of lauric acid (12:0) was less inhibited by fructose refeeding and by (+)-decanoyl-carnitine than the oxidation of oleic acid was, suggesting a peroxisomal beta-oxidation of lauric acid. Little lauric acid was esterified in triacylglycerol fraction, except at high substrate concentrations or in the fructose-refed state. With [1-14C]myristic acid (14:0), [1-14C]lauric acid (12:0), [1-14C]octanoic acid (8:0) and [2-14C]adrenic acid (22:4(n - 6] as substrate for hepatocytes from carbohydrate-refed rats, a large fraction of the 14C-labelled esterified fatty acids consisted of newly synthesized palmitic acid (16:0), stearic acid (18:0) and oleic acid (18:1) while intact [1-14C]oleic acid substrate was esterified directly. With [9,10-3H]myristic acid as the substrate, small amounts of shortened 3H-labelled beta-oxidation intermediates were found. With [U-14C]palmitic acid, no shortened fatty acids were detected. It was concluded that when the mitochondrial fatty acid oxidation is down-regulated such as in the carbohydrate-refed state, medium-chain fatty acids can partly be retailored to long-chain fatty acids by peroxisomal beta-oxidation followed by synthesis of C16 and C16 fatty acids which can then stored as triacylglycerol.     

Evidence for peroxisomal retroconversion of adrenic acid (22:4(n-6)) and docosahexaenoic acids (22:6(n-3)) in isolated liver cells

The intracellular localization of the oxidation of [2-14C]adrenic acid (22:4(n-6)) and [1-14C]docosahexaenoic acid (22:6(n-3)) was studied in isolated liver cells. The oxidation of 22:4(n-6) was 2-3-times more rapid than the oxidation of 22:6(n-3), [1-14C]arachidonic acid (20:4(n-6)) or [1-14C]oleic acid (18:1). (+)-Decanoylcarnitine and lactate, both known to inhibit mitochondrial beta-oxidation, reduced the oxidation of 18:1 distinctly more efficiently than with 22:4(n-6) and 22:6(n-3). In liver cells from rats fed a diet containing partially hydrogenated fish oil, the oxidation of 22:6(n-6) and 22:6(n-3) was increased by 30-40% compared with cells from rats fed a standard pellet diet. With 18:1 as substrate, the amount of fatty acid oxidized was very similar in cells from animals fed standard pellets or partially hydrogenated fish oil. Shortened fatty acids were not produced from [5,6,8,9,11,12,14,15-3H]arachidonic acid. In hepatocytes from rats starved and refed 20% fructose, a large fraction of 14C from 22:4 was recovered in 14C-labelled C14-C18 fatty acids. Oxidation of 22:4 thus caused a high specific activity of the extramitochondrial pool of acetyl-CoA. The results suggest that 22:4(n-6) and to some extent 22:6(n-3) are oxidized by peroxisomal beta-oxidation and by this are retroconverted to arachidonic acid and eicosapentaenoic acid.     

Incorporation into phospholipid classes and metabolism via desaturation and elongation of various 14C-labelled (n-3) and (n-6) polyunsaturated fatty acids in trout astrocytes in primary culture

The incorporation and metabolism of [1-14C]18:3(n-3), [1-14C]20:5(n-3), [1-14C]18:2(n-6), and [1-14C]20:4(n-6) were studied in primary cultures of trout brain astrocytes. There were no significant differences between the amounts of individual fatty acids incorporated into total lipid at 22 degrees C, with greater than 90% of all the fatty acids being incorporated into polar lipid classes. The distributions of 18:2(n-6), 18:3(n-3), and 20:5(n-3) in individual phospholipid classes at 22 degrees C were very similar, with 57-63 and 18-24% being incorporated into phosphatidylcholine and phosphatidylethanolamine, respectively. Approximately equal amounts of 20:4(n-6), approximately 30% of the total, were incorporated into each of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. The metabolism of the (n-3) fatty acids to longer-chain and more unsaturated species was significantly greater than that of (n-6) acids, but delta 4-desaturase activity was very low. A culture temperature of 10 degrees C increased the incorporation of all the fatty acids into total lipid and that of C20 fatty acids into polar lipid. At 10 degrees C, the incorporation of C20 fatty acids into phosphatidylethanolamine and phosphatidylinositol was increased, and the incorporation into phosphatidylcholine and phosphatidylserine was decreased. The distribution of C18 fatty acids was unchanged at the lower temperature, as was the desaturation and elongation of all the polyunsaturated fatty acids incorporated.     

Effects of 3-thia fatty acids on feed intake, growth, tissue fatty acid composition, beta-oxidation and Na+,K+-ATPase activity in Atlantic salmon

Atlantic salmon (Salmo salar) with an initial mass of 86 g were reared in 12 °C seawater for 8 weeks to a final average mass of 250 g. The fish were fed fish meal and fish oil-based diet supplemented with either 0%, 0.3% or 0.6% of tetradecylthioacetic acid (TTA), a 3-thia fatty acid. The specific growth rate (SGR) decreased with increasing dietary dose of TTA. The SGR of the group fed 0% of TTA (Control) was 1.8; that of the group fed 0.3% of TTA (TTA-L) was 1.7, and that of the group fed 0.6% of TTA (TTA-H) was 1.5. The mortality increased with increased dietary dose of TTA. The mitochondrial β-oxidation capacity in the liver of fish fed the TTA diets was 1.5 to 2 times higher than that of the Control fish. TTA supplementation caused substantial changes in the fatty acid compositions of the phospholipids (PL), triacylglycerols (TAG) and free fatty acids (FFA) of gills, heart and liver. The percentages of n−3 fatty acids, particularly 22:6 n−3, increased in fish fed diets containing TTA, while the percentage of the saturated FAs 14:0 and 16:0 in the PL fractions of the gills and heart decreased. The sum of monounsaturated FAs in the PL and TAG fractions from liver was significantly higher in fish fed diets containing TTA. TTA itself was primarily incorporated into PL. Two catabolic products of TTA (sulphoxides of TTA) were identified, and these products were particularly abundant in the kidney. TTA supplementation had no significant effect on the activity of the membrane-bound enzyme Na⁺,K⁺-ATPase.     

Partitioning of polyunsaturated fatty acid oxidation between mitochondria and peroxisomes in isolated rat hepatocytes studied by HPLC separation of oxidation products

The extent of mitochondrial and peroxisomal contribution to beta-oxidation of 18-, 20- and 24-carbon n-3 and n-6 polyunsaturated fatty acids (PUFAs) in intact rat hepatocytes is not fully clear. In this study, we analyzed radiolabeled acid soluble oxidation products by HPLC to identify mitochondrial and peroxisomal oxidation of 24:5n-3, 18- and 20-carbon n-3 and n-6 PUFAs. Mitochondrial fatty acid oxidation produced high levels of ketone bodies, tricarboxylic acid cycle intermediates and CO(2), while peroxisomal beta-oxidation released acetate. Inhibition of mitochondrial fatty acid oxidation with 2-tetradecylglycidic acid (TDGA), high amounts of [14C]acetate from oxidation of 24:5n-3, 18- and 20-carbon PUFAs were observed. In the absence of TDGA, high amounts of [14C]-labeled mitochondrial oxidation products were formed from oxidation of 24:5n-3, 18- and 20-carbon PUFAs. With 18:1n-9, high amounts of mitochondrial oxidation products were formed in the absence of TDGA, and TDGA strongly suppressed the oxidation of this fatty acid. Data of this study indicated that a shift in the partitioning from mitochondrial to peroxisomal oxidation differed for each individual fatty acid and is a specific property of 24:5n-3, 18- and 20-carbon n-3 and n-6 PUFAs.[14C]22:6n-3 was detected with [3-14C]24:5n-3, but not with [1-14C]24:5n-3 as the substrate, while [14C]16:0 was detected with [1-14C]24:5n-3, but not with [3-14C]24:5n-3 as the substrate. Furthermore, the amounts of 14CO(2) were similar when cells were incubated with [3-14C]24:5n-3 versus [1-14C]24:5n-3. These findings indicated that the proportion of 24:5n-3 oxidized in mitochondria was high, and that 24:5n-3 and 24:6n-3 were mostly beta-oxidized only one cycle in peroxisomes.     

Sex-related differences in desaturation and chain elongation of essential fatty acids studied in isolated rat hepatocytes

When [14C]linoleic acid (18:2(n-6)) or [14C]dihomogammalinolenic acid (20:3(n-6)) was incubated with isolated liver cells from rats fed an essential fatty acid deficient diet, delta 6- and delta 5-desaturation, chain elongation and synthesis of 14C-labelled C14-C18 fatty acids (from [14C]acetate) were enhanced in female cells compared with male ones. No sex difference in total secretion of very low density lipoproteins (VLDL) was observed. However, VLDL secreted from female cells contained significantly more C16-C18 fatty acids than male cells. It is suggested that the observed sex differences, at least in part, may be related to the different content of fatty acid binding proteins in female cells compared with males.     

Direct effects of temperature on phospholipid and polyunsaturated fatty acid metabolism in isolated brain cells from rainbow trout, Oncorhynchus mykiss.

1. The direct effects of temperature on the metabolism of [1-14C]18:2(n-6), [1-14C]18:3(n-3), [1-14C]20:4(n-6) and [1-14C]20:5(n-3) were studied in isolated brain cells from rainbow trout, Oncorhynchus mykiss. 2. Recovery of radioactivity from all the polyunsaturated fatty acids (PUFA) in total lipid was significantly greater at 5 and 15 degrees C than at 25 degrees C. 3. The lower incubation temperatures decreased the relative net incorporation of all the 14C-labelled PUFA into phosphatidylcholine (PC) and increased the relative incorporation of the PUFA into the other phosphoglycerides, especially phosphatidylethanolamine (PE). 4. The effects on PC were generally more significant between 25 and 15 degrees C, whereas the effects on PE were generally significant both between 25 and 15 degrees C and between 15 and 5 degrees C. 5. This suggests that the lysophospholipid acyltransferases responsible for the incorporation of PUFA into different phosphoglycerides may have differential sensitivities to temperature. 6. In contrast, the acyltransferase activities showed fatty acyl preferences that were independent of temperature. 7. Although a trend towards decreased activity at 5 degrees C was apparent, temperature generally had little significant effect on the relative percentages of the PUFA metabolized via the desaturase pathways.     

Mitochondrial and peroxisomal oxidation of arachidonic and eicosapentaenoic acid studied in isolated liver cells

The partitioning between peroxisomal and mitochondrial beta-oxidation of [1-14C]eicosapentaenoic acid (20:5(n-3] and [1-14C]arachidonic acid (20:4(n-6)) was studied. In hepatocytes from fasted rats approximately 70% of the fatty acid substrate was oxidized with oleic, linoleic, eicosapentaenoic and docosahexaenoic (22:6(n-3)) acid, even more with adrenic (22:4(n-6)) and less with arachidonic acid. When the mitochondrial oxidation was suppressed by fructose refeeding and by (+)-decanoylcarnitine, the fatty acid oxidation in per cent of that in cells from fasted rats was with 18:1(n-9) 7%, 18:2(n-6) 8%, 20:4(n-6) 12%, 20:5(n-3) 20%, 22:4(n-6) 57% and for 22:6(n-3) 29%. The fraction of 14C recovered in palmitate and other newly synthesized fatty acids after fructose refeeding decreased in the order 22:4(n-6) greater than 22:6(n-3) greater than 20:5(n-3) greater than 20:4(n-6) and was very small with 18:1(n-9) and 18:2(n-6). In cells from both fed and fructose-refed animals 20:5(n-3) was efficiently elongated to 22:5(n-3) and 22:6(n-3). 20:5(n-3) and 20:4(n-6) were not elongated after fasting. The phospholipid incorporation with [1-14C]20:5(n-3) decreased during prolonged incubations while it remained stable with [1-14C]arachidonic acid. The results suggest that peroxisomes contribute more to the oxidation of 20:5(n-3) than with 20:4(n-6) although both substrates are probably oxidized mainly in the mitochondria.     

Dietary omega-6 fatty acid lowering increases bioavailability of omega-3 polyunsaturated fatty acids in human plasma lipid pools

BackgroundDietary linoleic acid (LA, 18:2n-6) lowering in rats reduces n-6 polyunsaturated fatty acid (PUFA) plasma concentrations and increases n-3 PUFA (eicosapentaenoic (EPA) and docosahexaenoic acid (DHA)) concentrations.ObjectiveTo evaluate the extent to which 12 weeks of dietary n-6 PUFA lowering, with or without increased dietary n-3 PUFAs, alters unesterified and esterified plasma n-6 and n-3 PUFA concentrations in subjects with chronic headache.DesignSecondary analysis of a randomized trial. Subjects with chronic headache were randomized for 12 weeks to (1) average n-3, low n-6 (L6) diet; or (2) high n-3, low n-6 LA (H3–L6) diet. Esterified and unesterified plasma fatty acids were quantified at baseline (0 weeks) and after 12 weeks on a diet.ResultsCompared to baseline, the L6 diet reduced esterified plasma LA and increased esterified n-3 PUFA concentrations (nmol/ml), but did not significantly change plasma arachidonic acid (AA, 20:4n-6) concentration. In addition, unesterified EPA concentration was increased significantly among unesterified fatty acids. The H3–L6 diet decreased esterified LA and AA concentrations, and produced more marked increases in esterified and unesterified n-3 PUFA concentrations.ConclusionDietary n-6 PUFA lowering for 12 weeks significantly reduces LA and increases n-3 PUFA concentrations in plasma, without altering plasma AA concentration. A concurrent increase in dietary n-3 PUFAs for 12 weeks further increases n-3 PUFA plasma concentrations and reduces AA.     

Differences in the conversion of the polyunsaturated fatty acids [1-(14)C]22:4(n-6) and [1-(14)C]22:5(n-3) to [(14)C]22:5(n-6) and [(14)C]22:6(n-3) in isolated rat hepatocytes

The reasons why most cellular lipids preferentially accumulate 22:6(n-3) rather than 22:5(n-6) are poorly understood. In the present work the metabolisms of the precursor fatty acids, [1-(14)C]20:4(n-6), [1-(14)C]22:4(n-6) versus [1-(14)C]20:5(n-3), [1-(14)C]22:5(n-3) in isolated rat hepatocytes were compared. The addition of lactate and L-decanoylcarnitine increased the formation of [(14)C]24 fatty acid intermediates and the final products, [(14)C]22:5(n-6) and [(14)C]22:6(n-3). In the absence of lactate and L-decanoylcarnitine, no [(14)C]24 fatty acids and [(14)C]22:5(n-6) were detected when [1-(14)C]22:4(n-6) was the substrate, whereas small amounts of the added [1-(14)C]22:5(n-3) was converted to [(14)C]22:6(n-3). Lactate reduced the oxidation of [1-(14)C]22:4(n-6) and [1-(14)C]22:5(n-3) while L-decanoylcarnitine did not. No significant differences between the total oxidation or esterification of the two substrates were observed. By fasting and fructose refeeding the amounts of [(14)C]24:4(n-6) and [(14)C]24:5(n-3) were increased by 2.5- and 4-fold, respectively. However, the levels of [(14)C]22:5(n-6) and [(14)C]22:6(n-3) were similar in hepatocytes from fasted and refed versus fed rats. With hepatocytes from rats fed a fat free diet the levels of [(14)C]24 fatty acid intermediates were low while the further conversion of the n-6 and n-3 substrates was high and more equal, approx. 33% of [1-(14)C]22:4(n-6) was converted to [(14)C]22:5(n-6) and 43% of [1-(14)C]22:5(n-3) was converted to [(14)C]22:6(n-3). The moderate differences found in the conversion of [1-(14)C]22:4(n-6) versus [1-(14)C]22:5(n-3) to [(14)C]22:5(n-6) and [(14)C]22:6(n-3), respectively, and the equal rates of oxidation of the two substrates could thus not explain the abundance of 22:6(n-3) versus the near absence of 22:5(n-6) in cellular membranes.     

Effects of water temperature and diets containing palm oil on fatty acid desaturation and oxidation in hepatocytes and intestinal enterocytes of rainbow trout (Oncorhynchus mykiss)

Food grade fisheries have reached their sustainable limits while aquaculture production has increased to meet consumer demands. However, for growth in aquaculture to continue and utilise sustainable, feeding ingredients, alternatives to fish oil (FO), the predominant lipid component of fish diets, must be developed. Therefore, there is currently considerable interest in the regulation of fatty acid metabolism in fish in order to determine strategies for the best use of plant oils in diets for commercially important cultured fish species. Plant oils are characteristically rich in C18 polyunsaturated fatty acids (PUFA) but devoid of C20 and C22 highly unsaturated fatty acids (HUFA) found in FO. The fatty acyl desaturase enzyme activities involved in the biosynthesis of HUFA from PUFA are known to be under nutritional regulation and can be increased in fish fed diets rich in plant oils. However, fatty acid desaturase activity is also known to be modulated by water temperature in fish. The present study aimed to investigate the interaction between water temperature and diet in the regulation of fatty acid metabolism in rainbow trout. Trout, acclimatized to 7, 11 or 15 degrees C, were fed for 4 weeks on diets in which the FO was replaced in a graded manner by palm oil. At the end of the trial, fatty acyl desaturation/elongation and beta-oxidation activities were determined in isolated hepatocytes and intestinal enterocytes using [1-14C]18:3n-3 as substrate, and samples of liver were collected for analysis of lipid and fatty acid composition. The most obvious effect of temperature was that fatty acid desaturation/elongation and beta-oxidation were reduced in both hepatocytes and intestinal enterocytes from fish maintained at the highest water temperature (15 degrees C). There were differences between the two tissues with the highest desaturation/elongation and beta-oxidation activities tending to be in fish held at 11 degrees C in the case of hepatocytes, but 7 degrees C in enterocytes. Correlations between fatty acid metabolism and dietary palm oil were most clearly observed in desaturation/elongation activities in both hepatocytes and enterocytes at 11 degrees C. The highest beta-oxidation activities were generally observed in fish fed FO alone in both hepatocytes and enterocytes with palm oil having differential effects in the two cell types.     

Effects of essential fatty acid deficiency and supplementation with docosahexaenoic acid (DHA; 22:6n-3) on cellular fatty acid compositions and fatty acyl desaturation in a cell culture model

The desaturation of [1-(14)C] 18:3n-3 to docosahexaenoic acid (DHA; 22:6n-3) is enhanced in an essential fatty acid deficient cell line (EPC-EFAD) in comparison with the parent cell line (EPC) from carp. In the present study, the effects of DHA on lipid and fatty acid compositions, and the metabolism of [1-(14)C]18:3n-3 were investigated in EPC-EFAD cells in comparison with EPC cells. DHA supplementation had only relatively minor effects on lipid content and lipid class compositions in both EPC and EPC-EFAD cells, but significantly increased the amount of DHA, 22:5n-3, eicosapentaenoic acid (EPA; 20:5n-3), total n-3 polyunsaturated fatty acids (PUFA), total PUFA and saturated fatty acids in total lipid and total polar lipid in both cell lines. Retroconversion of supplemental DHA to EPA was significantly greater in EPC cells. Monounsaturated fatty acids, n-9 and n-6PUFA were all decreased in total lipid and total polar lipid in both cell lines by DHA supplementation. The incorporation of [1-(14)C]18:3n-3 was greater into EPC-EFAD compared to EPC cells but DHA had no effect on the incorporation of [1-(14)C]18:3n-3 in either cell line. In contrast, the conversion of [1-(14)C]18:3n-3 to tetraenes, pentaenes and total desaturation products was similar in the two cell lines and was significantly reduced by DHA supplementation in both cell lines. However, the production of DHA from [1-(14)C]18:3n-3 was significantly greater in EPC-EFAD cells compared to EPC cells and, whereas DHA supplementation had no effect on the production of DHA from [1-(14)C]18:3n-3 in EPC cells, DHA supplementation significantly reduced the production of DHA from [1-(14)C] 18:3n-3 in EPC-EFAD cells. Greater production of DHA in EPC-EFAD cells could be a direct result of significantly lower levels of end-product DHA in these cells' lipids compared to EPC cells. Consistent with this, the suppression of DHA production upon DHA supplementation was associated with increased cellular and membrane DHA concentrations in EPC-EFAD cells. However, an increase in cellular DHA content to similar levels failed to suppress DHA production in DHA-supplemented EPC cells. A possible explanation is that greatly increased levels of EPA, derived from retroconversion of the added DHA, acts to offset the suppression of the pathway by DHA by stimulating conversion of EPA to DHA in DHA-supplemented EPC cells.     

Selective incorporation of various C-22 polyunsaturated fatty acids in Ehrlich ascites tumor cells

Three 14C-labeled 22-carbon polyunsaturated fatty acids, 7,10,13,16-[14C]docosatetraenoic acid (22:4(n-6)), 7,10,13,16,19-[14C]docosapentaenoic acid (22:5(n-3)), and 4,7,10,13,16,19-[14C]docosahexaenoic acid (22:6(n-3)), were compared with [3H]arachidonic acid (20:4(n-6] and [14C]linoleic acid (18:2(n-6)) to characterize their incorporation into the lipids of Ehrlich ascites cells. The relatively rapid incorporation of the labeled 22-carbon acids into phosphatidic acid indicated that substantial amounts of these acids may be incorporated through the de novo pathway of phospholipid synthesis. In marked contrast to 20:4(n-6), the 22-carbon acids were incorporated much less into choline glycerophospholipids (CGP) and inositol glycerophospholipids (IGP). No selective preference was apparent for the (n-3) or (n-6) type of fatty acids. The amounts of the acids incorporated into diacylglycerophosphoethanolamine were in the order of: 22:6(n-3) greater than 20:4(n-6) much greater than 22:5(n-3) greater than or equal to 22:4(n-6) greater than 18:2(n-6), whereas for alkylacylglycerophosphoethanolamine they were in the order of: 22:4(n-6) greater than 22:6(n-3) greater than 22:5(n-3) much greater than 20:4(n-6) greater than 18:2(n-6). Of the mechanisms possibly responsible for the selective entry of 22-carbon acids into ethanolamine glycerophospholipids, the most reasonable explanation was that the cytidine-mediated ethanolamine phosphotransferase may have a unique double selectivity: for hexaenoic species of diacylglycerol and for 22-carbon polyunsaturated fatty acid-containing species of alkylacylglycerol. The relative distribution of fatty acids between newly incorporated and already maintained lipid classes suggested that IGP may function in Ehrlich cells as an intermediate pool for the retention of polyunsaturated fatty acids in glycerolipids.     

Dietary LC-PUFA and environmental salinity modulate the fatty acid biosynthesis capacity of the euryhaline teleost thicklip grey mullet (Chelon labrosus)

The capacity to biosynthesise long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) depends upon the complement and function of key enzymes commonly known as fatty acyl desaturases and elongases. The presence of a Δ5/Δ6 desaturase enabling the biosynthesis of docosahexaenoic acid (22:6n-3, DHA) through the “Sprecher pathway” has been reported in Chelon labrosus. Research in other teleosts have demonstrated that LC-PUFA biosynthesis can be modulated by diet and ambient salinity. The present study aimed to assess the combined effects of partial dietary replacement of fish oil (FO) by vegetable oil (VO) and reduced ambient salinity (35 ppt vs 20 ppt) on the fatty acid composition of muscle, enterocytes and hepatocytes of C. labrosus juveniles. Moreover, the enzymatic activity over radiolabelled [1-¹⁴C] 18:3n-3 (α-linolenic acid, ALA) and [1-¹⁴C] 20:5n-3 (eicosapentaenoic acid, EPA) to biosynthesise n-3 LC-PUFA in hepatocytes and enterocytes, and the gene regulation of the C. labrosus fatty acid desaturase-2 (fads2) and elongation of very long chain fatty acids protein 5 (elovl5) in liver and intestine was also investigated. Recovery of radiolabelled products including stearidonic acid (18:4n-3, SDA), 20:5n-3, tetracosahexaenoic acid (24:6n-3, THA) and 22:6n-3 in all treatments except FO35-fish, provided compelling evidence that a complete pathway enabling the biosynthesis of EPA and DHA from ALA is present and active in C. labrosus. Low salinity conditions upregulated fads2 in hepatocytes and elovl5 in both cell types, regardless of dietary composition. Interestingly, FO20-fish showed the highest amount of n-3 LC-PUFA in muscle, while no differences in VO-fish reared at both salinities were found. These results demonstrate a compensatory capacity of C. labrosus to biosynthesise n-3 LC-PUFA under reduced dietary supply, and emphasise the potential of low salinity conditions to stimulate this pathway in euryhaline fish.     

Fatty acid utilisation and metabolism in caecal enterocytes of rainbow trout (Oncorhynchus mykiss) fed dietary fish or copepod oil

A combined fatty acid metabolism assay was employed to determine fatty acid uptake and relative utilisation in enterocytes isolated from the pyloric caeca of rainbow trout. In addition, the effect of a diet high in long-chain monoenoic fatty alcohols present as wax esters in oil derived from Calanus finmarchicus, compared to a standard fish oil diet, on caecal enterocyte fatty acid metabolism was investigated. The diets were fed for 8 weeks before caecal enterocytes from each dietary group were isolated and incubated with [1-14C]fatty acids: 16:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:1n-9, 20:4n-6, 20:5n-3, and 22:6n-3. Uptake was measured over 2 h with relative utilisation of different [1-14C]fatty acids calculated as a percentage of uptake. Differences in uptake were observed, with 18:1n-9 and 18:2n-6 showing the highest rates. Esterification into cellular lipids was highest with 16:0 and C18 fatty acids, accounting for over one-third of total uptake, through predominant incorporation in triacylglycerol (TAG). The overall utilisation of fatty acids in phospholipid synthesis was low, but highest with 16:0, the most prevalent fatty acid recovered in intracellular phosphatidylcholine (PC) and phosphatidylinositol (PI), although exported PC exhibited higher proportions of C20/C22 polyunsaturated fatty acids (PUFA). Other than 16:0, incorporation into PC and PI was highest with C20/C22 PUFA and 20:4n-6 respectively. Recovery of labelled 18:1n-9 in exported TAG was 3-fold greater than any other fatty acid which could be due to multiple esterification on the glycerol 'backbone' and/or increased export. Approximately 20-40% of fatty acids taken up were beta-oxidised, and was highest with 20:4n-6. Oxidation of 20:5n-3 and 22:6n-3 was also surprisingly high, although 22:6n-3 oxidation was mainly attributed to retroconversion to 20:5n-3. Metabolic modification of fatty acids by elongation-desaturation was generally low at <10% of [1-14C]fatty acid uptake. Dietary copepod oil had generally little effect on fatty acid metabolism in enterocytes, although it stimulated the elongation and desaturation of 16:0 and elongation of 18:1n-9, with radioactivity recovered in longer n-9 monoenes. The monoenoic fatty acid, 20:1n-9, abundant in copepod oil as the homologous alcohol, was poorly utilised with 80% of uptake remaining unesterified in the enterocyte. However, the fatty acid composition of pyloric caeca was not influenced by dietary copepod oil.     

Effect of diets enriched in Delta6 desaturated fatty acids (18:3n-6 and 18:4n-3), on growth, fatty acid composition and highly unsaturated fatty acid synthesis in two populations of Arctic charr (Salvelinus alpinus L.)

This study aimed to test the hypothesis that diets containing relatively high amounts of the Delta6 desaturated fatty acids stearidonic acid (STA, 18:4n-3) and gamma-linolenic acid (GLA, 18:3n-6), may be beneficial in salmonid culture. The rationale being that STA and GLA would be better substrates for highly unsaturated fatty acid (HUFA) synthesis as their conversion does not require the activity of the reputed rate-limiting enzyme, fatty acid Delta6 desaturase. Duplicate groups of two Arctic charr (Salvelinus alpinus L.) populations with different feeding habits, that had been reported previously to show differences in HUFA biosynthetic capacity, were fed for 16 weeks on two fish meal based diets containing 47% protein and 21% lipid differing only in the added lipid component, which was either fish oil (FO) or echium oil (EO). Dietary EO had no detrimental effect on growth performance and feed efficiency, mortalities, or liver and flesh lipid contents in either population. The proportions of 18:2n-6, 18:3n-3, 18:3n-6, 18:4n-3, 20:3n-6 and 20:4n-3 in total lipid in both liver and flesh were increased by dietary EO in both populations. However, the percentages of 20:5n-3 and 22:6n-3 were reduced by EO in both liver and flesh in both strains, whereas 20:4n-6 was only significantly reduced in flesh. In fish fed FO, HUFA synthesis from both [1-(14)C]18:3n-3 and [1-(14)C]20:5n-3 was significantly higher in the planktonivorous Coulin charr compared to the demersal, piscivorous Rannoch charr morph. However, HUFA synthesis was increased by EO in Rannoch charr, but not in Coulin charr. In conclusion, dietary EO had differential effects in the two populations of charr, with HUFA synthesis only stimulated by EO in the piscivorous Rannoch morph, which showed lower activities in fish fed FO. However, the hypothesis was not proved as, irrespective of the activity of the HUFA synthesis pathway in either population, feeding EO resulted in decreased tissue levels of n-3HUFA and 20:4n-6. This has been observed previously in salmonids fed vegetable oils, and thus the increased levels of Delta6 desaturated fatty acids in EO did not effectively compensate for the lack of dietary HUFA.     

Reversibility of the changes induced by n-3 fatty acids in mouse plasma, liver and blood cell lipids

The changes induced by dietary n-3 fatty acids (FA) in the lipids and FA of plasma, liver and blood cells, and their reversibility, was studied in mice given a diet containing 9% fish oil (FO) for 2 weeks and then returned to, and kept for another 2 weeks on, the usual standard lab chow diet. In plasma, the concentrations of phospholipids (PL), mostly phosphatidylcholine (PC), triacylglycerols (TG), cholesterol and cholesterol esters (CE) decreased rapidly after starting the FO diet, and remained low from day 3 onwards. This decrease was concomitant with a remarkable reduction in the n-6 FA, especially 18:2n-6, not compensated for by the relative enrichment in n-3 FA induced by FO. In liver, TG and CE decreased and PL slightly increased, all of them showing reduced n-6/n-3 ratios. Sphingomyelin, which lacks polyunsaturated FA other than small amounts of 18:2 and 24:2n-6, showed altered ratios between its very long chain monoenes and saturates. In the washout phase, the most rapid event was an immediate increase in 18:2n-6 and after a few days in 20:4n-6 in plasma and liver, where most of the lipid and FA changes were reversed completely in about 10 days. In the case of blood cells even 2 weeks were insufficient for a reversal to the initial n-6/n-3 ratios. The lipid class responsible for this lack of reversibility was phosphatidylethanolamine, PC having returned to the initial fatty acid composition during the stated period.